Current methods for differentiating isolates of predominant lineages of pathogenic bacteria often do not provide sufficient resolution to define precise relationships. Here, we describe a high-throughput genomics approach that provides a high-resolution view of the epidemiology and microevolution of a dominant strain of methicillin-resistant Staphylococcus aureus (MRSA). This approach reveals the global geographic structure within the lineage, its intercontinental transmission through four decades, and the potential to trace person-to-person transmission within a hospital environment. The ability to interrogate and resolve bacterial populations is applicable to a range of infectious diseases, as well as microbial ecology.

BackgroundSeveral studies have addressed the epidemiology of community-associated Staphylococcus aureus (CA-SA) in Europe; nonetheless, a comprehensive perspective remains unclear. In this study, we aimed to describe the population structure of CA-SA and to shed light on the origin of methicillin-resistant S. aureus (MRSA) in this continent.Methods and FindingsA total of 568 colonization and infection isolates, comprising both MRSA and methicillin-susceptible S. aureus (MSSA), were recovered in 16 European countries, from community and community-onset infections. The genetic background of isolates was characterized by molecular typing techniques (spa typing, pulsed-field gel electrophoresis and multilocus sequence typing) and the presence of PVL and ACME was tested by PCR. MRSA were further characterized by SCCmec typing. We found that 59% of all isolates were associated with community-associated clones. Most MRSA were related with USA300 (ST8-IVa and variants) (40%), followed by the European clone (ST80-IVc and derivatives) (28%) and the Taiwan clone (ST59-IVa and related clonal types) (15%). A total of 83% of MRSA carried Panton-Valentine leukocidin (PVL) and 14% carried the arginine catabolic mobile element (ACME). Surprisingly, we found a high genetic diversity among MRSA clonal types (ST-SCCmec), Simpson’s index of diversity = 0.852 (0.788–0.916). Specifically, about half of the isolates carried novel associations between genetic background and SCCmec. Analysis by BURP showed that some CA-MSSA and CA-MRSA isolates were highly related, suggesting a probable local acquisition/loss of SCCmec.ConclusionsOur results imply that CA-MRSA origin, epidemiology and population structure in Europe is very dissimilar from that of USA.

CO-releasing molecules (CO-RMs) were previously shown by us to be more potent bactericides than CO gas. This suggests a mechanism of action for CO-RM, which either potentiates the activity of CO or uses another CO-RM-specific effect. We have also reported that CORM-2 induces the expression of genes related to oxidative stress. In the present study we intend to establish whether the generation of reactive oxygen species by CO-RMs may indeed result in the inhibition of bacterial cellular function. We now report that two CO-RMs (CORM-2 and ALF062) stimulate the production of ROS in Escherichia coli, an effect that is abolished by addition of antioxidants. Furthermore, deletion of genes encoding E. coli systems involved in reactive oxygen species scavenging, namely catalases and superoxide dismutases, potentiates the lethality of CORM-2 due to an increase of intracellular ROS content. CORM-2 also induces the expression of the E. coli DNA repair/SOS system recA, and its inactivation enhances toxicity of CORM-2. Moreover, fluorescence microscopy images reveal that CORM-2 causes DNA lesions to bacterial cells. We also demonstrate that cells treated with CORM-2 contain higher levels of free iron arising from destruction of iron-sulfur proteins. Importantly, we show that CO-RMs generate hydroxyl radicals in a cell-free solution, a process that is abolished by scavenging CO. Altogether, we provide a novel insight into the molecular basis of CO-RMs action by showing that their bactericidal properties are linked to cell damage inflicted by the oxidative stress that they are able to generate.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of infection in the community (CA-MRSA), but in spite of its relevance, no data exist concerning its epidemiology in Portugal. In this study, we aimed to evaluate the prevalence, population structure, and origin of MRSA in the Portuguese community. A total of 527 isolates, both methicillin-susceptible S. aureus (MSSA) and MRSA, were collected from individuals with no healthcare-related risk factors attending 16 healthcare institutions in Portugal. Isolates were characterized for the presence of mecA, Panton-Valentine leukocidin (PVL), and arginine catabolic mobile element (ACME), and by staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), spa, and multilocus sequence typing (MLST). Susceptibility to a panel of 13 antibiotics was tested. Isolates relatedness was analyzed by goeBURST and BURP. We found a high frequency (21.6%) of MRSA in the community. However, only 11.4% of the isolates belonged to typical CA-MRSA epidemic clones (USA300, USA400, USA700, Southwest Pacific, European, and ST398). The remaining isolates, which constituted the great majority (88.6%), belonged to hospital-associated MRSA (HA-MRSA) epidemic clones, namely, to the EMRSA-15 clone (77.2%). PVL was rare and carried by 17 isolates only (five MRSA and 12 MSSA). In the whole collection, some MRSA and MSSA were highly related. The high frequency of MRSA in the community in Portugal seems to result mainly from dissemination from the hospital. They might also have emerged from an extant MSSA population, by SCCmec acquisition, or MRSA clonal introduction from abroad.